1. Field of the Invention
This invention relates to a naphthoquinone alkylating agent that contains an oligonucleotide probe. In one embodiment the alkylating agent may be activated by light irradiation, and in another embodiment the crosslinking agent may be activated by enzymatic or chemical reduction.
2. Background of the Related Art
Currently prescribed chemotherapeutic agents acting at the level of DNA are often effective, but their therapeutic index is quite poor, limited by the lack of target specificity. An international research effort has been underway using a wide range of techniques to develop a gene specific drug--a "magic bullet" that is aimed not at an individual organism or cell type, but a single DNA sequence within a cell.
The technological advances allowing for facile DNA synthesis have produced innumerable protocols which rely on custom oligonucleotides, used as probes to screen for complementary sequences within plasmids, chromosomes and DNA libraries. See for example, Landegren et al., "DNA Diagnostics-Molecular Techniques and Automation", Science, 242, 229 (1988). The specificity of oligonucleotide hydridization has been utilized for "antisense" methods controlling selective expression of genes both in vivo and in vitro. For example, see Miller et al., "Oligonucleotide Inhibitors of Gene Expression in Living Cells: New Opportunities in Drug Design", Ann. Reports in Med. Chem., 23, 295 (1988). Sequence recognition by the binding of probes most often depends on only the non-covalent forces of hydrogen bonding formed between complementary base pairs. Complexation of this type is quite sufficient for many applications, but covalent stabilization of duplex structures could simplify many of the current protocols and provide new opportunities for processing DNA in a sequence specific manner.
Only recently introduced, the technique of oligonucleotide-directed irreversible DNA modification holds great potential as an in vitro tool for molecular biologists. See for example, Dervan, "Design of Sequence-Specific DNA-Binding Molecules", Science, 232, 464-471 (1986); and Iverson et al, in "Nonenzymatic Sequence-Specific Cleavage of Single-Stranded DNA to Nucleotide Resolution. DNA Methyl Thiolether Probes", J. Am. Chem. Soc., 109, 1241-1243 (1987). Site specificity is enforced by the hybridization of the oligomer-reactant to its complement sequence prior to drug action. Target selectivity can then be conferred, in theory, to most reactive compounds by attaching them to oligonucleotides. The required prehybridization step, however, generally limits this technique's applicability to accessible single strand polynucleotide targets or duplex probes when triple helical formation is possible, see Maher III et al., "Inhibition of DNA Binding Proteins by Oligonucleotide-Directed Triple Helix Formation", Science, 245, 725-730 (1989).
Site-directed covalent modification is also constrained by the nature of the reactive group incorporated into the oligomer. In vivo application of this method has not been demonstrated and must await the development of new reactive compounds that are compatible with a cellular environment.
Recently, messenger RNA has become a viable target for inhibiting the expression of a desired gene in vivo. See for example, Toulme et al., "Antimessenger oligodeoxyribonucleotides: an alternative to antisense RNA for artificial regulation of gene expression--a review", Gene, 72, 51-58 (1988); and, Stein et al., "Oligodeoxyribonucleotides as Inhibitors of Gene Expression : A Review", Cancer Research, 48, 2659-2668 (1988). Compounds created for this selective reaction have drawn from the advances in site specific modification of DNA, for example see: Barton, "Metals and DNA: Molecular Left-Handed Complements", Science, 233, 727-734 (1986), and Dervan, in Science, 232 (1986) supra. Use of such compounds also depends on the synthesis of metabolically stable oligonucleotides that can transverse cell membranes. For example, see: Blake et al., "Hybridization Arrest of Globin Synthesis in Rabbit Reticulocyte Lysates and Cells by Oligodeoxyribonucleoside Methylphosphonates", Biochemistry, 24, 6139-6145 (1985). Also, see Agrawal et al., "Oligodeoxynucleoside phosphoramidates and phosphorothioates as inhibitors of human immunodeficiency virus", Proc. Nat' l. Acad. Sci. U.S.A., 85, 7079-7083 (1988).
Although a large number of reactive appendages are available for related use in vitro, as reported by Iverson et al., J. Am. Chem. Soc., 109 (1987) supra: and by Dervan, Science, 232 (1986) supra, only a limited set of these may be incorporated into techniques for in vivo use. See Mori et al., in "Oligodeoxynucleotide Analogs with 5+-linked anthraquinone", FEBS Letters. 249, 213-218 (1989) who reported the synthesis of a 5'-linked oligodeoxynucleotide in which a covalently attached group links the nucleotide to an anthraquinone molecule. The anthraquinone molecule was chosen for its potential radical-producing moiety, that does not necessarily require the presence of a metal ion or optical activation. Such a molecule, however, is incapable of alkylating the target DNA. Also, the anthroquinone probably is not photochemically active, due to the resonance structures of all of the carbon-carbon double bonds.
Wagner et al. have reported that methyl naphthoquinone sensitizes the selective oxidation of thymine, in "Photo-Oxidation of Thymine Sensitized by 2-Methyl-1,4-Naphthoquinone: Analysis of Products Including Three Novel Photo-Dimers", Photochem. Photobiol., 40, 589-597, (1984).
In an article by Antonini et al., entitled "2- and 6-Methyl-1,4-naphthoquinone Derivatives as Potential Bioreductive Alkylating Agents", J. Med. Chem., 25, 730-735 (1982). A number of antineoplastic agents which possess both a quinone nucleus and a substituent that permits them to function as bioreductive alkylating agents. Antonini et al. reported the synthesis of a series of 2-and 6-methyl-1,4-naphthoquinone derivatives, and evaluated them for antitumor effects on mice bearing Sarcoma 180 ascites cells. These antitumor agents were thought to be activated by reduction to an alkylating species. Such reduction was believed to take place due to enzymes produced by the metabolic system of hypoxic tumor cells, functioning under low oxygen tension. Antonini et al. did not, however, describe or suggest the delivery of the naphthoquinone derivatives into the cell DNA by using a target specific probe. Nor did Antonini et al. describe the modification or use of such naphthoquinones as UV activated alkylating agents. Indeed, as shown in Example 4, the structure shown in Scheme 1 of Antonini et al., with their --CH.sub.2 --X group on the quinone ring structure would not be practical, since it would attack and degrade a probe during the linking process. Additionally, Antonini et al. inset their --CH.sub.2 --X group on carbon 6 of the benzene ring, they do not, however, suggest inserting the --CH.sub.2 --X group on the other positions of the benzene ring.
Yabusaki et al., in PCT Published Application No. WO 85/02628 describe cross-linking agents for binding an oligonucleotide probe to a target DNA or RNA molecule. Three types of cross-linking agents are described, including "bi-functional photoreagents", "mixed chemical and biochemical bifunctional reagents" and "bifunctional chemical cross-linking molecules". The bifunctional photoreagents contain two photochemically reactive sites that bind covalently to the probe and to the target molecules. The mixed chemical and photochemical bifunctional reagent is bound non-photochemically to the probe molecule, followed by photochemical binding to the target molecule. Non-photochemical binding is described as a chemical reaction such as alkylation, condensation or additional. Bifunctional chemical cross-linking molecules are said to be activated either catalytically or by high temperature following hybridization.
Although Yabusaki et al. generally hypothesize the concept of a bifunctional photochemical reagent and a mixed chemical and photochemical reagent, there is no specific description of these molecules. All of the reagents they describe are well known photochemical reagents, these include the psoralen derivatives, including furocoumarins, the benzodipyrone derivatives, and the bis-azide derivatives. None of these molecules, however, work on the basis of reductive activation. In addition, these compounds are not practical in vivo reagents. It would be difficult or nearly impossible to photoactivate these reagents in vivo. These reagents, especially the psoralen derivatives are toxic, causing severe burning of the organism after exposure to sunlight. Finally, the covalent crosslinks formed by psoralens are not permanent, rather, they are degraded by UV irradiation.
Two recent articles reported the use or psoralen crosslinks of DNA substrates, the first by Van Houten et al., in "Action mechanism of ABC excision nuclease on a DNA substrate containing a psoralen crosslink at a defined position", Proc. Nat'l. Acad. Sci. USA, 83, 8077-8081 (1986), and the second by Lee et al., in "Interaction of Psoralen-Derivatized Oligodeoxyribonucleoside Methyl-phosphonates with Single-Stranded DNA", Biochemistry, 27, 3197-3203 (1988). Both articles reported covalent crosslinking between the DNA molecule and a complementary oligomer that contains a psoralen derivative. The covalent binding of the psoralen derivative to the DNA molecule was activated by UV irradiation. Accordingly, just like the Yabusaki patent application, none of these references describe the chemically activated covalent binding agent which may be used in vivo. Additionally, as discussed above, the covalent crosslinks, formed by psoralens are not permanent, being degraded by UV irradiation.
The techniques of Northern and Southern blotting are two of the most powerful and frequently used procedures in molecular biology, see Wall et al., "Northern and Southern Blots", Methods Enz., 152, 572-573 (1987). Yet the necessary manipulations are time consuming and are not likely to be automated under current technology. Often the polynucleotide (RNA, DNA) under analysis must first be fractionate by size, transferred onto a solid support and then treated through a series of steps to ensure only specific binding of a probe. Detection of the hybridized products usually depends on radiolabelling, heavy metal derivatization or antibody complexation. The methods of blotting have been a staple of basic research, and now also serve in an ever increasing number of commercial kits used to diagnose genetic malignant and infectious diseases (see Landegren et al. Science, 242, (1988) supra). Related advances have also allowed these processes to aid in forensic science, see Higuchi et al., "DNA typing from single hairs", Nature, 332, 543-546 (1988); and, the Human Genome Project, see Conner et al., "Detection of sickle cell .beta..sup.s -globin allele by hybridization with synthetic oligonucleotides", Proc. Nat'l. Acad. Sci. USA, 80, 278-282 (1983).
Psoralens have been used to randomly crosslink duplex DNA during hybridization in order to facilitate Southern Blotting procedures. This new test is referred to as Reverse Southern blotting. For example, see Gamper et al., in "Reverse Southern Hybridization", Nucl. Acids Res., 14, 9943 (1986). Other biochemical and reduction activated reagents are needed to replace or complement psoralens for sequence detection and to provide an alternate set of conditions for duplex stabilization.
Accordingly, none of the related art describes or suggests using UV and/or visible light ("UV/Vis.") activation with a naphthoquinone derivative in order to permanently alkylate a biological molecule such as DNA.
Therefore, it is an object of the present invention to provide a new class of photochemically activated alkylating probes which form a permanent covalent crosslink.
Another object of the present invention is to provide an enzymatic or chemical reduction activated alkylating probe which can be used in vivo.
A further object of the present invention is to provide a new class of photochemical and reduction activated Reverse Southern blotting reagents for conjugating and permanently crosslinking target oligonucleotides and facilitate blotting procedures, sequence detection and scission.